Plant glucose-6-phosphate translocator

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a glucose-6-phosphate/phosphate translocator. The invention also relates to the construction of a chimeric gene encoding all or a portion of the glucose-6-phosphate/phosphate translocator, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the glucose-6-phosphate/phosphate translocator in a transformed host cell.

This application claims priority benefit to U.S. Provisional ApplicationNo. 60/107,910 filed Nov. 10, 1998, now abandoned.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingglucose-6-phosphate/phosphate translocators in plants and seeds.

BACKGROUND OF THE INVENTION

During C₃ photosynthesis, energy from solar radiation is used for theformation of phosphorylated C3 sugar phosphates, triose phosphates(triosP), and 3-phosphoglycerate (3-PGA). These products are thenexported from the chloroplasts into the cytosol via thetrioseP/3-PGA/phosphate translocator (TPT). In the mature leaves of mostplants, the exported photosynthates are then used in the formation ofsucrose, which is allocated via the phloem to the heterotrophic plantorgans, such as young leaves, roots, seeds, fruits or tubers. In thesesink tissues, sucrose serves as a source of carbon and energy and isfirst cleaved by the action of invertases or sucrose synthase. Theproducts of these reactions are converted into hexose phosphates.

Nongreen plastids of heterotrophic tissues import carbohydrates to fuelmetabolism in those cells. Amyloplasts, found in storage tissues,convert the carbohydrates into starch which is efficiently stored. Ingeneral, the plastids of heterotrophic tissues cannot generate hexosephosphates from C3 compounds due to an absence of fructose1,6-bisphosphatase activity. Thus, the TPTs that transported thetriose-phosphates out of the chloroplasts in photosynthetic tissues arenot useful for transporting them back into the storage plastids ofheterotrophic tissues. Therefore, nongreen plastids rely on the importof hexose phosphates to supply the materials for starch biosynthesis,and for generating energy through the oxidative pentose phosphatepathway. A hexose-phosphate/phosphate translocator (or transporter, orantiporter, which are used interchangeably herein) allows membranepassage of hexose-phosphate while simultaneously transporting inorganicphosphate, or triose-phosphates, in the opposite direction. In nongreenplant tissues glucose 6-phosphate (Glc6P) is the preferred hexosephosphate taken up by nonphotosynthetic plastids. The translocationevent is selective, as shown by the inability of amyloplast membranes totransport phosphoenolpyruvate, fructose-6-phosphate, orglucose-1-phosphate. It has been shown recently (Kammerer, B. et al.(1998) The Plant Cell 10:105-117) that Glc6P is taken up by nongreenplastids via a glucose-6-phosphate/phosphate translocator (GP/PT).Glucose-6-phosphate imported by the GP/PT protein can be incorporatedinto starch, releasing inorganic phosphate, or can serve as a substratefor the oxidative pentose phosphate pathway, yielding triose phosphates.

GP/PT is one of the main translocators to provide plastids with carbonfor biosynthetic pathways and energy. GP/PT transcripts are abundant inheterotrophic tissues active in starch synthesis, such as potato tubers,maize kernels, and pea roots (Kammerer et al. (1998) Plant Cell10:105-117), but are barely detectable in photosynthetic tissues.However, endosperm-specific starch synthesis, found in maize and barley,occurs in the cytosol and does not utilize GP/PT transport (Denyer etal. (1996) Plant Physiol 112:779-185; Thorbjornsen et al. (1996) Plant J10:243-250). So not all starch containing tissues are necessarilydependent upon GP/PT activities. Still, it is clear that GP/PTs play anintegral role in maintaining starch synthesis and energy metabolism innormal plant growth and development. Accordingly, the availability ofnucleic acid sequences encoding all or a portion of the GP/PT proteinwould facilitate studies to better understand hexose phosphatetransport, provide genetic tools for the manipulation of biosyntheticpathways and energy production, and may provide possible targets forherbicides or the engineering of herbicide resistance.

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides comprising anucleotide sequence encoding a first polypeptide of at least 401 aminoacids that has at least 70% identity based on the Clustal method ofalignment when compared to a rice glucose-6-phosphate/phosphatetranslocator (GP/PT) polypeptide of SEQ ID NO:2, a nucleotide sequenceencoding a first polypeptide of at least 395 amino acids that has atleast 86% identity based on the Clustal method of alignment whencompared to a soybean GP/PT polypeptide of SEQ ID NO:4, and a nucleotidesequence encoding a first polypeptide of at least 302 amino acids thathas at least 93% identity based on the Clustal method of alignment whencompared to a wheat GP/PT polypeptide of SEQ ID NO:6. The presentinvention also relates to an isolated polynucleotide comprising thecomplement of the nucleotide sequences described above.

It is preferred that the isolated polynucleotides of the claimedinvention consists of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 3, and 5, that codes for the polypeptideselected from the group consisting of SEQ ID NOs:2, 4, and 6. Thepresent invention also relates to an isolated polynucleotide comprisinga nucleotide sequences of at least one of 60 (preferably at least one of40, most preferably at least 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, and 5, and the complement of such nucleotide sequences.

The present invention relates to a chimeric gene comprising an isolatedpolynucleotide of the present invention operably linked to suitableregulatory sequences.

The present invention relates to an isolated host cell comprising achimeric gene of the present invention or an isolated polynucleotide ofthe present invention. The host cell may be eukaryotic, such as a yeastor a plant cell, or prokaryotic, such as a bacterial cell. The presentinvention also relates to a virus, preferably a baculovirus, comprisingan isolated polynucleotide of the present invention or a chimeric geneof the present invention.

The present invention relates to a process for producing an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention, the process comprisingeither transforming or transfecting an isolated compatible host cellwith a chimeric gene or isolated polynucleotide of the presentinvention.

The present invention relates to a GP/PT polypeptide of at least 401amino acids that has at least 70% identity based on the Clustal methodof alignment when compared to a glucose-6-phosphate/phosphatetranslocator polypeptide of SEQ ID NO:2, a GP/PT polypeptide of at least395 amino acids that has at least 86% identity based on the Clustalmethod of alignment when compared to a glucose-6-phosphate/phosphatetranslocator polypeptide of SEQ ID NO:4, and a composition consisting ofa GP/PTpolypeptide of at least 302 amino acids that has at least 93%identity based on the Clustal method of alignment when compared to aglucose-6-phosphate/phosphate translocator polypeptide of SEQ ID NO:6.

The present invention relates to a method of selecting an isolatedpolynucleotide that affects the level of expression of a GP/PTpolypeptide in a host cell, preferably a plant cell, the methodcomprising the steps of:

constructing an isolated polynucleotide of the present invention or anisolated chimeric gene of the present invention;

introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell;

measuring the level a GP/PT polypeptide in the host cell containing theisolated polynucleotide; and

comparing the level of a GP/PT polypeptide in the host cell containingthe isolated polynucleotide with the level of a GP/PT polypeptide in ahost cell that does not contain the isolated polynucleotide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a GP/PT polypeptide gene,preferably a plant GP/PT polypeptide gene, comprising the steps of:synthesizing an oligonucleotide primer comprising a nucleotide sequenceof at least one of 60 (preferably at least one of 40, most preferably atleast one of 30) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:1, 3, and 5and the complement of such nucleotide sequences; and amplifying anucleic acid fragment (preferably a cDNA inserted in a cloning vector)using the oligonucleotide primer. The amplified nucleic acid fragmentpreferably will encode a portion of a GP/PT amino acid sequence.

The present invention also relates to a method of obtaining a nucleicacid fragment encoding all or a substantial portion of the amino acidsequence encoding a GP/PT polypeptide comprising the steps of: probing acDNA or genomic library with an isolated polynucleotide of the presentinvention; identifying a DNA clone that hybridizes with an isolatedpolynucleotide of the present invention; isolating the identified DNAclone; and sequencing the cDNA or genomic fragment that comprises theisolated DNA clone.

A further embodiment of the instant invention is a method for evaluatingat least one compound for its ability to inhibit the activity of aGP/PT, the method comprising the steps of: (a) transforming a host cellwith a chimeric gene comprising a nucleic acid fragment encoding aGP/PT, operably linked to suitable regulatory sequences; (b) growing thetransformed host cell under conditions that are suitable for expressionof the chimeric gene wherein expression of the chimeric gene results inproduction of GP/PT in the transformed host cell; (c) optionallypurifying the GP/PT expressed by the transformed host cell; (d) treatingthe GP/PT with a compound to be tested; and (e) comparing the activityof the GP/PT that has been treated with a test compound to the activityof an untreated GP/PT, thereby selecting compounds with potential forinhibitory activity.

The present invention relates to a composition comprising an isolatedpolynucleotide of the present invention.

The present invention relates to an isolated polynucleotide comprisingat least one of 30 contiguous nucleotides of a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5 and thecomplement of such sequences.

The present invention relates to an expression cassette comprising anisolated polynucleotide of the present invention operably linked to apromoter.

The present invention relates to a method for positive selection of atransformed cell comprising:

(a) transforming a plant cell with a chimeric gene of claim 5 or anexpression cassette of the present invention; and

(b) growing the transformed plant cell, wherein the plant cell is amonocot or a dicot such as rice, soybean or wheat, under conditionsallowing expression of the polynucleotide in an amount sufficient tocomplement a glucose-6-phosphate/phosphate translocator auxotroph toprovide a positive selection means.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A and 1B show a comparison of the amino acid sequences of therice (r10n.pk0016.f6:fis), soybean (soycon, a contig of sequences listedin Table 3), and wheat (wr1.pk0053.e6) GP/PT (SEQ ID NOs:2, 4, and 6)versus the pea (SEQ ID NO:7) and the corn (SEQ ID NO:8) proteins. Therice amino acid sequence is presented from the first methionine in thesequence, however there is an in-frame 33 amino acid leader attached tothis polypeptide (amino acids 1-33 of SEQ ID NO:2). The wheat sequencerepresents a truncated cDNA clone that starts approximately at theamino-terminal end of the mature protein.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Glucose-6-Phosphate/Phosphate Translocators SEQ ID NO: (Nucleo-(Amino Protein Clone Designation tide) Acid) Rice GP/PT (Oryza sativa)r10n.pk0016.f6:fis 1 2 Soybean GPIPT (Glycine Contig of: 3 4 max)s2.07c09 sde4c.pk0001.g4 sdp4c.pk003.o9 se4.pk0005.e7 sgs3c.pk001.i5sl1.pk0058.a8 sl2.pk0047.b3 sls2c.pk013.b10 sr1.pk0007.d2 src3c.pk020.e1Wheat GP/PT (Triticum sp.) wr1.pk0053.e6:fis 5 6

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “polynucleotide” is a nucleotide sequence such as anucleic acid fragment. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, or synthetic DNA. An isolated polynucleotide of the presentinvention may include at least one of 60 contiguous nucleotides,preferably at least one of 40 contiguous nucleotides, most preferablyone of at least 30 contiguous nucleotides, of the nucleic acid sequenceof the SEQ ID NOs:1, 3, and 5.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, and 5, and the complement of such nucleotide sequences may beused in methods of selecting an isolated polynucleotide that affects theexpression of a polypeptide in a plant cell. A method of selecting anisolated polynucleotide that affects the level of expression of apolypeptide (such as GP/PT polypeptide) in a host cell (eukaryotic, suchas plant or yeast, prokaryotic such as bacterial, or viral) may comprisethe steps of: constructing an isolated polynucleotide of the presentinvention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide in the host cell containing the isolated polynucleotide withthe level of a polypeptide in a host cell that does not contain theisolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6× SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2× SSC, 0.5% SDS at 45° C.for 30 min, and then repeated twice with 0.2× SSC, 0.5% SDS at 50° C.for 30 min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2× SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1× SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least 70% identical,preferably at least 80% identical to the amino acid sequences reportedherein. Preferred nucleic acid fragments encode amino acid sequencesthat are at least 85% identical to the amino acid sequences reportedherein. More preferred nucleic acid fragments encode amino acidsequences that are at least 90% identical to the amino acid sequencesreported herein. Most preferred are nucleic acid fragments that encodeamino acid sequences that are at least 95% identical to the amino acidsequences reported herein. Suitable nucleic acid fragments not only havethe above homologies but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids. Sequence alignments andpercent identity calculations were performed using the Megalign programof the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison,Wis.). Multiple alignment of the sequences was performed using theClustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Default parameters for pairwise alignments using the Clustal method wereKTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of severalglucose-6-phosphate/phosphate translocator have been isolated andidentified by comparison of random plant cDNA sequences to publicdatabases containing nucleotide and protein sequences using the BLASTalgorithms well known to those skilled in the art. The nucleic acidfragments of the instant invention may be used to isolate cDNAs andgenes encoding homologous proteins from the same or other plant species.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, methods of nucleic acid hybridization, and methodsof DNA and RNA amplification as exemplified by various uses of nucleicacid amplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other GP/PT, either as cDNAs or genomicDNAs, could be isolated directly by using all or a portion of theinstant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, the entire sequences canbe used directly to synthesize DNA probes by methods known to theskilled artisan such as random primer DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 60 (preferably one of at least 40, most preferably one ofat least 30) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, and 5 and thecomplement of such nucleotide sequences may be used in such methods toobtain a nucleic acid fragment encoding a substantial portion of anamino acid sequence of a polypeptide. The present invention relates to amethod of obtaining a nucleic acid fragment encoding a substantialportion of a polypeptide of a gene (such as GP/PT) preferably asubstantial portion of a plant polypeptide of a gene, comprising thesteps of: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least one of 60 (preferably at least one of 40, mostpreferably at least one of 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, and 5, and the complement of such nucleotide sequences; andamplifying a nucleic acid fragment (preferably a cDNA inserted in acloning vector) using the oligonucleotide primer. The amplified nucleicacid fragment preferably will encode a portion of a polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of sugars, starches, and metabolites in thosecells.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded glucose-6-phosphate/phosphate translocator. An example of avector for high level expression of the instant polypeptides in abacterial host is provided (Example 6).

Additionally, the instant polypeptides can be used as a targets tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze the transport ofglucose-6-phosphate into the plastid. Accordingly, inhibition of theactivity of one or more of the enzymes described herein could lead toinhibition of plant growth. Thus, the instant polypeptides could beappropriate for new herbicide discovery and design.

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various rice, soybean, or corntissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Rice, Soybean, and Wheat Library TissueClone r10n Rice 15 day old leaf* r10n.pk0016.f6:fis s2 Soybean Seed, 19Days After Flowering s2.07c09 sde4c Soybean Developing Embryo (9-11 mm)sde4c.pk0001.g4 sdp4c Soybean Developing Pods (10-12 mm) sdp4c.pk003.o9se4 Soybean embryo, 19 days after flowering se4.pk0005.e7 sgs3c SoybeanSeeds 25 Hours After Germ- sgs3c.pk001.i5 ination sl1 SoybeanTwo-Week-Old Developing sl1.pk0058.a8 Seedlings sl2 Soybean Two-Week-OldDeveloping sl2.pk0047.b3 Seedlings Treated With 2.5 ppm chlorimuronsls2c Soybean Infected With Sclerotinia sls2c.pk013.b10 sclerotiorummycelium sr1 Soybean Root sr1.pk0007.d2 src3c Soybean 8 Day Old RootInfected With src3c.pk020.e1 Cyst Nematode wr1 Wheat root from 7 day oldseedling wr1.pk0053.e6:fis *This library was normalized essentially asdescribed in U.S. Pat. No. 5,482,845, incorporated hereby by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding glucose-6-phosphate/phosphate translocators wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3

Characterization of cDNA Clones Encoding Glucose-6-Phosphate/PhosphateTranslocator

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to GP/PTfrom pea and corn (NCBI Accession No. gi 2997591 and gi 2997589,respectively). Shown in Table 3 are the BLAST results for individualESTs (“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), contigs assembled from two or more ESTs(“Contig”), contigs assembled from an FIS and one or more ESTs(“Contig*”), or sequences encoding the entire protein derived from anFIS, a contig, or an FIS and PCR (“CGS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toGP/PT BLAST pLog Score Clone Status 2997591 r10n.pk0016.f6:fis FIS155.00 Contig of: Contig 254.00 s2.07c09 sde4c.pk0001.g4 sdp4c.pk003.o9se4.pk0005.e7 sgs3c.pk001.i5 sl1.pk0058.a8 sl2.pk0047.b3 sls2c.pk013.b10sr1.pk0007.d2 src3c.pk020.e1 BLAST pLog Score Clone Status 2997589wr1.pk0053.e6:fis FIS 162.00

FIG. 1 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:2, 4, and 6, and the pea sequence (SEQ ID NO:7), and the cornsequence (SEQ ID NO:8). The data in Table 4 represents a calculation ofthe percent identity of the amino acid sequences set forth in SEQ IDNOs:2, 4, and the pea sequence (SEQ ID NO:7), and the amino acidsequences set forth in SEQ ID NO:6 and the corn sequence (SEQ ID NO:8).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toGP/PT Percent Identity to SEQ ID NO. 2997591 2 67.3% 4 85.3% PercentIdentity to SEQ ID NO. 2997589 6 92.7%

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a GP/PT. These sequences represent thefirst rice, soybean, and wheat sequences encoding GP/PT.

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML 103. Plasmid pML 103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the P subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/1 μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 7 Evaluating Compounds for Their Ability to Inhibit the Activityof Glucose-6-Phosphate/Phosphate Translocators

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 6, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptides may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instant polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for GP/PT are presented by Kammerer et al.(1998) Plant Cell 10:105-117.

8 1 1661 DNA Oryza sativa 1 gcacgagctt acaaaagaag gaaaaatgaa tttaatgggcctgattcgag cttgtgcatc 60 cgtgtaatta cactatattg tcggattgaa gaagactcgatgcgagcggt ctctatggcc 120 gcaacccatc gccgccatag acgtacggac ggccggctacaacacaacac agtaactgta 180 actgaagaag ccggcgccac cattcccgcc gccaatatgctcgccgccgc cgcctccgtc 240 aagctctcca cggccgggac cgccacggcg cccaagatggcattattcaa gcccctccac 300 ctccctcccc tcttcgccgc cgccgccgcc gccgccgggccacgtcccct ctccctctcg 360 gcgcgccccc tgtaccggca gcaggacccg ttattcctggcgtcgcgcgt cgcgtcaccg 420 gcgccgccgc cgccgtccgc caccgccgac ggcgcccggccggtggaggc tgcaccggcg 480 ggcgcggcgc cggaggaggc ggcgaggcgg gccaagatcggggtctactt cgcgacgtgg 540 tgggcgctga acgtgatctt caacatctac aacaagaaggtgctcaacgc gttcccttac 600 ccgtggctca cgtccacgct ctccctcgcc gccggctccgccatcatgct cgcctcctgg 660 gccaccagga tcgccgaggc gcccgccacc gacctcgatttctggaaggc cctgtcaccg 720 gtggcgatcg cgcacaccat cgggcacgtg gcggcgacggtgagcatggc gaaggtggcg 780 gtgtcgttca cccacatcat caagagcggc gagccggcgttcagcgtgct cgtctccagg 840 ttcttcctcg gcgagcactt cccggcgccg gtctacttctccctcctccc aatcatcggc 900 ggatgcgccc tcgccgccat caccgagctc aatttcaacatgattggatt catgggggcg 960 atgatctcga acctggcatt cgtgttccgg aacatattctccaagaaggg gatgaagggc 1020 aagtcggtga gcgggatgaa ctactacgcc tgcctctccatgctctcgct ggtcatcctc 1080 ctccctttcg ccttcgccat ggaggggccc aaggtgtgggctgccggctg gcaaaaagcc 1140 gtcgccgaga tcggtccaaa cttcgtctgg tgggtggcggcgcagagtgt gttctaccac 1200 ttgtacaacc aagtgtccta catgtcattg gacgagatctcaccactgac attcagcatc 1260 ggcaacacca tgaagcggat ctccgtcatt gtcgcctccatcatcatctt ccacacgccg 1320 gtccagccca tcaacgccct cggagccgcc attgcgattctcggaacttt catctactct 1380 caggcgaagc agtaatggca taagcatgga aatggcacccgttttcgcca caagtcgcga 1440 ctcggtgctc gggatgtaac aatgctatga gcatatatccaaggatccct tgtaatatat 1500 tagatcttct tttcttttca tttttctttc tctaggtgtagctgccaatg tttcttctga 1560 attcgtcgag ttcttgtact atgactatga tgatgtagagaggtggatct gcctaaataa 1620 agctgttaga ttttttgcta aaaaaaaaaa aaaaaaaaaa a1661 2 464 PRT Oryza sativa 2 Ala Arg Ala Tyr Lys Arg Arg Lys Asn GluPhe Asn Gly Pro Asp Ser 1 5 10 15 Ser Leu Cys Ile Arg Val Ile Thr LeuTyr Cys Arg Ile Glu Glu Asp 20 25 30 Ser Met Arg Ala Val Ser Met Ala AlaThr His Arg Arg His Arg Arg 35 40 45 Thr Asp Gly Arg Leu Gln His Asn ThrVal Thr Val Thr Glu Glu Ala 50 55 60 Gly Ala Thr Ile Pro Ala Ala Asn MetLeu Ala Ala Ala Ala Ser Val 65 70 75 80 Lys Leu Ser Thr Ala Gly Thr AlaThr Ala Pro Lys Met Ala Leu Phe 85 90 95 Lys Pro Leu His Leu Pro Pro LeuPhe Ala Ala Ala Ala Ala Ala Ala 100 105 110 Gly Pro Arg Pro Leu Ser LeuSer Ala Arg Pro Leu Tyr Arg Gln Gln 115 120 125 Asp Pro Leu Phe Leu AlaSer Arg Val Ala Ser Pro Ala Pro Pro Pro 130 135 140 Pro Ser Ala Thr AlaAsp Gly Ala Arg Pro Val Glu Ala Ala Pro Ala 145 150 155 160 Gly Ala AlaPro Glu Glu Ala Ala Arg Arg Ala Lys Ile Gly Val Tyr 165 170 175 Phe AlaThr Trp Trp Ala Leu Asn Val Ile Phe Asn Ile Tyr Asn Lys 180 185 190 LysVal Leu Asn Ala Phe Pro Tyr Pro Trp Leu Thr Ser Thr Leu Ser 195 200 205Leu Ala Ala Gly Ser Ala Ile Met Leu Ala Ser Trp Ala Thr Arg Ile 210 215220 Ala Glu Ala Pro Ala Thr Asp Leu Asp Phe Trp Lys Ala Leu Ser Pro 225230 235 240 Val Ala Ile Ala His Thr Ile Gly His Val Ala Ala Thr Val SerMet 245 250 255 Ala Lys Val Ala Val Ser Phe Thr His Ile Ile Lys Ser GlyGlu Pro 260 265 270 Ala Phe Ser Val Leu Val Ser Arg Phe Phe Leu Gly GluHis Phe Pro 275 280 285 Ala Pro Val Tyr Phe Ser Leu Leu Pro Ile Ile GlyGly Cys Ala Leu 290 295 300 Ala Ala Ile Thr Glu Leu Asn Phe Asn Met IleGly Phe Met Gly Ala 305 310 315 320 Met Ile Ser Asn Leu Ala Phe Val PheArg Asn Ile Phe Ser Lys Lys 325 330 335 Gly Met Lys Gly Lys Ser Val SerGly Met Asn Tyr Tyr Ala Cys Leu 340 345 350 Ser Met Leu Ser Leu Val IleLeu Leu Pro Phe Ala Phe Ala Met Glu 355 360 365 Gly Pro Lys Val Trp AlaAla Gly Trp Gln Lys Ala Val Ala Glu Ile 370 375 380 Gly Pro Asn Phe ValTrp Trp Val Ala Ala Gln Ser Val Phe Tyr His 385 390 395 400 Leu Tyr AsnGln Val Ser Tyr Met Ser Leu Asp Glu Ile Ser Pro Leu 405 410 415 Thr PheSer Ile Gly Asn Thr Met Lys Arg Ile Ser Val Ile Val Ala 420 425 430 SerIle Ile Ile Phe His Thr Pro Val Gln Pro Ile Asn Ala Leu Gly 435 440 445Ala Ala Ile Ala Ile Leu Gly Thr Phe Ile Tyr Ser Gln Ala Lys Gln 450 455460 3 1815 DNA Glycine max unsure (1687) unsure (1701) unsure (1716)unsure (1808) 3 ctcgtgccgc tcattgctgt tcaccatttt caattcattc ctgtgttagattccaagtgg 60 tttggtttgg tttgacataa ataaaccttt caagatctca gaggggcagaagatcaaagc 120 caagacaaca cactctcata tataaaagag tgtctctgat aatttgatctttgtttcttc 180 tctttctctc ctttgagatt tttcagcacc ccaccaaggt tttgtccgaaaagcttgaat 240 ttttcgccta ccctttacaa tgatctcttc attgagacaa cctgttgtagggatcagtgg 300 ttctgatctt cttttgaggc aaagacatgc aaccctaatt aaggcaaggtcctttttacc 360 ctctttgtca agagaaaagg gtcaaagatc tcttgtttca gttcaaaagccacttcacat 420 tgctgcttct cttggtgttg gaaattttgt gtcagtgaag agtgatgatgacaaaagggg 480 tgatttggtg aagtgtgagg cctatgaagc agacagatca gaggttgagggtgcaagcac 540 accatcagaa gctgcaaaga aggtgaaaat tgggatatat tttgcaacctggtgggcttt 600 gaatgtggtg ttcaatattt ataacaagaa ggttttgaat gcttacccttacccttggct 660 tacttcaact ctctcacttg catgtgggtc tcttatgatg ttgatctcttgggccactgg 720 gattgctgaa gccccaaaga ctgatcctga gttttggaag tctttgttccctgttgctgt 780 tgcacataca attggacatg tagcggcaac agttagcatg tcaaaagttgcggtatcatt 840 cacacacatt atcaagagtg gtgaacctgc ttttagtgtt ctggtttcaagatttctttt 900 gggtgagagc tttcctgtgc cagtctatct gtctttaatt ccaatcattggtggatgtgc 960 acttgctgct gtgactgagc tcaatttcaa tatgatcggt tttatgggggccatgatatc 1020 gaatttggca tttgtattcc gtaatatctt ttcgaaaaag ggcatgaagggaaagtctgt 1080 tagtggaatg aattactacg catgtttatc tattttatct cttgctattctcacaccctt 1140 cgcaattgct gtggaaggac cgcaaatgtg ggctgctgga tggcaaacagccatgtctca 1200 aattggaccc caattcatat ggtggctagc tgctcaaagt gtattctatcatctatacaa 1260 tcaagtgtca tacatgtctc tggatcagat ctctcctttg acgtttagcattggaaacac 1320 catgaaacgt atatctgtca ttgtgtcttc cattattatc ttccacacaccagttcaacc 1380 catcaatgct cttggtgccg ccattgctat ccttggaacc ttcttgtattcacaggcgaa 1440 actatagagt tggggaacta agattcctct agggacttgt ttgttacagttcttcatgct 1500 caaccagcga caaggaagtt gattttgtaa tgtgtcactg ctggaattttgtaatactag 1560 gatgatctgg ctatcagtcc atctgtagat ataataataa aaataatgctcctgaggaaa 1620 cattatcata atacagatta ttagggtttt tttttttttt tgaatttctgaaatgccaat 1680 gatttgnaaa taaaatgccc ntttggttta taatangtaa tgaagaaagaatttctcaag 1740 acattggtta ctataaaggg agccttgcaa taaaagtgat gcctaatgatcaaatttgat 1800 gctccacnca cacac 1815 4 395 PRT Glycine max 4 Met IleSer Ser Leu Arg Gln Pro Val Val Gly Ile Ser Gly Ser Asp 1 5 10 15 LeuLeu Leu Arg Gln Arg His Ala Thr Leu Ile Lys Ala Arg Ser Phe 20 25 30 LeuPro Ser Leu Ser Arg Glu Lys Gly Gln Arg Ser Leu Val Ser Val 35 40 45 GlnLys Pro Leu His Ile Ala Ala Ser Leu Gly Val Gly Asn Phe Val 50 55 60 SerVal Lys Ser Asp Asp Asp Lys Arg Gly Asp Leu Val Lys Cys Glu 65 70 75 80Ala Tyr Glu Ala Asp Arg Ser Glu Val Glu Gly Ala Ser Thr Pro Ser 85 90 95Glu Ala Ala Lys Lys Val Lys Ile Gly Ile Tyr Phe Ala Thr Trp Trp 100 105110 Ala Leu Asn Val Val Phe Asn Ile Tyr Asn Lys Lys Val Leu Asn Ala 115120 125 Tyr Pro Tyr Pro Trp Leu Thr Ser Thr Leu Ser Leu Ala Cys Gly Ser130 135 140 Leu Met Met Leu Ile Ser Trp Ala Thr Gly Ile Ala Glu Ala ProLys 145 150 155 160 Thr Asp Pro Glu Phe Trp Lys Ser Leu Phe Pro Val AlaVal Ala His 165 170 175 Thr Ile Gly His Val Ala Ala Thr Val Ser Met SerLys Val Ala Val 180 185 190 Ser Phe Thr His Ile Ile Lys Ser Gly Glu ProAla Phe Ser Val Leu 195 200 205 Val Ser Arg Phe Leu Leu Gly Glu Ser PhePro Val Pro Val Tyr Leu 210 215 220 Ser Leu Ile Pro Ile Ile Gly Gly CysAla Leu Ala Ala Val Thr Glu 225 230 235 240 Leu Asn Phe Asn Met Ile GlyPhe Met Gly Ala Met Ile Ser Asn Leu 245 250 255 Ala Phe Val Phe Arg AsnIle Phe Ser Lys Lys Gly Met Lys Gly Lys 260 265 270 Ser Val Ser Gly MetAsn Tyr Tyr Ala Cys Leu Ser Ile Leu Ser Leu 275 280 285 Ala Ile Leu ThrPro Phe Ala Ile Ala Val Glu Gly Pro Gln Met Trp 290 295 300 Ala Ala GlyTrp Gln Thr Ala Met Ser Gln Ile Gly Pro Gln Phe Ile 305 310 315 320 TrpTrp Leu Ala Ala Gln Ser Val Phe Tyr His Leu Tyr Asn Gln Val 325 330 335Ser Tyr Met Ser Leu Asp Gln Ile Ser Pro Leu Thr Phe Ser Ile Gly 340 345350 Asn Thr Met Lys Arg Ile Ser Val Ile Val Ser Ser Ile Ile Ile Phe 355360 365 His Thr Pro Val Gln Pro Ile Asn Ala Leu Gly Ala Ala Ile Ala Ile370 375 380 Leu Gly Thr Phe Leu Tyr Ser Gln Ala Lys Leu 385 390 395 51281 DNA Triticum aestivum 5 gcacgagctc agaagcagca caaaagttga agatctcaatctattttgcg acttggtggg 60 cgcttaatgt gatctttaac atctataaca agaaggttctcaatgctttc ccgtatccct 120 ggctcacctc cacactatcc ctcgcctgtg gctcagcgatgatgctcttc tcatgggtca 180 cctgcctagt tgaggccccc aagactgact tagatttctggaaagcactc ttcccggttg 240 ctgtggctca tacaattgga catgttgctg ccacagtgagcatgtcaaag gtggcagtgt 300 cattcacaca cattatcaag agtgctgagc ctgcattcagtgttttggtg tcaaggttca 360 ttctcggaga gtcatttccg atgcctgtat atctttctcttctcccgatc attggtggtt 420 gtggtctagc tgctgcgaca gagctgaact ttaatatgattggatttatg ggtgccatga 480 tatcgaacct tgcatttgtt ttccgcaaca tcttctcgaagcggggcatg aaagggaagt 540 ctgtcagtgg catgaattac tacgcttgcc tttcaattatgtccctgatc atactcgcac 600 catttgctat tgctatggaa ggcccccaaa tgtgggccgctggatggcaa aaggctcttg 660 cagatgttgg ccccaacgtt ctctggtgga ttggtgcacagagcgttttc taccacctgt 720 ataaccaggt gtcctacatg tctttggacc agatttctccattgacgttt agcattggca 780 atacaatgaa gcgcatatca gttattgttt cgtcaatcattatcttccgt acacctgtcc 840 gccctgtaaa tgcactagga gctgccattg ccatctttggcacattcctg tactctcagg 900 caaagcagtg aggtttaaac tattgttgaa ggtcaggttcatggacgaag aatgtctgca 960 gaaatagtac caagcagtgg ggcaaatttt gcttgtgcaagtgtcttgta gatataggtt 1020 taggttttca tgcgagaggc gataataata atggaccatgttgctgtttc cttgttattt 1080 gttaaatcgc aaactgaact ccagtggcca tgaactgtcgttgtatcctg aaataagaac 1140 tgcatggaaa ttcgtctgac agttccgttg ctggaaagtaacgatagccc tgtgcttaac 1200 ggtaaattat tgatatttaa gaactttgtg ttaaaaaaaaaaaaaaaaaa aaaaaaaaaa 1260 aaaaaaaaaa aaaaaaaaaa a 1281 6 302 PRTTriticum aestivum 6 Thr Ser Ser Glu Ala Ala Gln Lys Leu Lys Ile Ser IleTyr Phe Ala 1 5 10 15 Thr Trp Trp Ala Leu Asn Val Ile Phe Asn Ile TyrAsn Lys Lys Val 20 25 30 Leu Asn Ala Phe Pro Tyr Pro Trp Leu Thr Ser ThrLeu Ser Leu Ala 35 40 45 Cys Gly Ser Ala Met Met Leu Phe Ser Trp Val ThrCys Leu Val Glu 50 55 60 Ala Pro Lys Thr Asp Leu Asp Phe Trp Lys Ala LeuPhe Pro Val Ala 65 70 75 80 Val Ala His Thr Ile Gly His Val Ala Ala ThrVal Ser Met Ser Lys 85 90 95 Val Ala Val Ser Phe Thr His Ile Ile Lys SerAla Glu Pro Ala Phe 100 105 110 Ser Val Leu Val Ser Arg Phe Ile Leu GlyGlu Ser Phe Pro Met Pro 115 120 125 Val Tyr Leu Ser Leu Leu Pro Ile IleGly Gly Cys Gly Leu Ala Ala 130 135 140 Ala Thr Glu Leu Asn Phe Asn MetIle Gly Phe Met Gly Ala Met Ile 145 150 155 160 Ser Asn Leu Ala Phe ValPhe Arg Asn Ile Phe Ser Lys Arg Gly Met 165 170 175 Lys Gly Lys Ser ValSer Gly Met Asn Tyr Tyr Ala Cys Leu Ser Ile 180 185 190 Met Ser Leu IleIle Leu Ala Pro Phe Ala Ile Ala Met Glu Gly Pro 195 200 205 Gln Met TrpAla Ala Gly Trp Gln Lys Ala Leu Ala Asp Val Gly Pro 210 215 220 Asn ValLeu Trp Trp Ile Gly Ala Gln Ser Val Phe Tyr His Leu Tyr 225 230 235 240Asn Gln Val Ser Tyr Met Ser Leu Asp Gln Ile Ser Pro Leu Thr Phe 245 250255 Ser Ile Gly Asn Thr Met Lys Arg Ile Ser Val Ile Val Ser Ser Ile 260265 270 Ile Ile Phe Arg Thr Pro Val Arg Pro Val Asn Ala Leu Gly Ala Ala275 280 285 Ile Ala Ile Phe Gly Thr Phe Leu Tyr Ser Gln Ala Lys Gln 290295 300 7 401 PRT Pisum sativum 7 Met Ile Ser Ser Leu Arg Gln Pro SerIle Ser Ile Ser Gly Ser Asp 1 5 10 15 Val Val Leu Arg Lys Arg His AlaThr Leu Ile Gln Leu Arg Pro Gln 20 25 30 Ser Phe Ser Pro Phe Ser Ser ArgGlu Lys Ser Gln Arg Ser Val Val 35 40 45 Ser Thr Lys Lys Pro Leu His LeuAla Cys Leu Gly Val Gly Asn Phe 50 55 60 Gly Ser Val Lys Asn Phe Glu SerGlu Ala Ser Phe Gly Gln Ser Asp 65 70 75 80 Leu Val Lys Cys Gly Ala TyrGlu Ala Asp Arg Ser Glu Val Glu Gly 85 90 95 Gly Asp Gly Thr Pro Ser GluAla Ala Lys Lys Val Lys Ile Gly Ile 100 105 110 Tyr Phe Ala Thr Trp TrpAla Leu Asn Val Val Phe Asn Ile Tyr Asn 115 120 125 Lys Lys Val Leu AsnAla Tyr Pro Tyr Pro Trp Leu Thr Ser Thr Leu 130 135 140 Ser Leu Ala CysGly Ser Leu Met Met Leu Ile Ser Trp Ala Thr Arg 145 150 155 160 Ile AlaGlu Ala Pro Lys Thr Asp Leu Glu Phe Trp Lys Thr Leu Phe 165 170 175 ProVal Ala Val Ala His Thr Ile Gly His Val Ala Ala Thr Val Ser 180 185 190Met Ser Lys Val Ala Val Ser Phe Thr His Ile Ile Lys Ser Gly Glu 195 200205 Pro Ala Phe Ser Val Leu Val Ser Arg Phe Ile Leu Gly Glu Thr Phe 210215 220 Pro Val Pro Val Tyr Leu Ser Leu Leu Pro Ile Ile Gly Gly Cys Ala225 230 235 240 Leu Ala Ala Val Thr Glu Leu Asn Phe Asn Met Ile Gly PheMet Gly 245 250 255 Ala Met Ile Ser Asn Leu Ala Phe Val Phe Arg Asn IlePhe Ser Lys 260 265 270 Lys Gly Met Lys Gly Lys Ser Val Ser Gly Met AsnTyr Tyr Ala Cys 275 280 285 Leu Ser Ile Leu Ser Leu Ala Ile Leu Thr ProPhe Ala Ile Ala Val 290 295 300 Glu Gly Pro Ala Met Trp Ala Ala Gly TrpGln Thr Ala Leu Ser Glu 305 310 315 320 Ile Gly Pro Gln Phe Ile Trp TrpVal Ala Ala Gln Ser Ile Phe Tyr 325 330 335 His Leu Tyr Asn Gln Val SerTyr Met Ser Leu Asp Glu Ile Ser Pro 340 345 350 Leu Thr Phe Ser Ile GlyAsn Thr Met Lys Arg Ile Ser Val Ile Val 355 360 365 Ser Ser Ile Ile IlePhe His Thr Pro Ile Gln Pro Val Asn Ala Leu 370 375 380 Gly Ala Ala IleAla Val Phe Gly Thr Phe Leu Tyr Ser Gln Ala Lys 385 390 395 400 Gln 8387 PRT Zea mays 8 Met Ile Pro Ser Val Arg Leu Ser Pro Gly Pro Ala AlaPhe Ser Gly 1 5 10 15 Ser Ser Leu Arg Ser Lys Leu Pro Ser Ile Pro SerIle Ser Ser Leu 20 25 30 Lys Pro Ser Lys Tyr Val Val Ser Ser Leu Lys ProLeu Tyr Leu Ala 35 40 45 Pro Leu Asp Gly Pro Arg Thr Ala Glu Leu Lys SerArg Arg Gln Pro 50 55 60 Leu Glu Phe Arg Cys Ser Ala Ser Ala Ala Asp AspLys Glu Ser Lys 65 70 75 80 Thr Gln Val Val Pro Val Gln Ser Glu Gly AlaGln Arg Leu Lys Ile 85 90 95 Ser Ile Tyr Phe Ala Thr Trp Trp Ala Leu AsnVal Ile Phe Asn Ile 100 105 110 Tyr Asn Lys Lys Val Leu Asn Ala Phe ProTyr Pro Trp Leu Thr Ser 115 120 125 Thr Leu Ser Leu Ala Cys Gly Ser AlaMet Met Leu Phe Ser Trp Ala 130 135 140 Thr Arg Leu Val Glu Ala Pro LysThr Asp Leu Asp Phe Trp Lys Val 145 150 155 160 Leu Phe Pro Val Ala ValAla His Thr Ile Gly His Val Ala Ala Thr 165 170 175 Val Ser Met Ser LysVal Ala Val Ser Phe Thr His Ile Ile Lys Ser 180 185 190 Ala Glu Pro AlaPhe Ser Val Leu Val Ser Arg Phe Phe Leu Gly Glu 195 200 205 Thr Phe ProIle Pro Val Tyr Leu Ser Leu Leu Pro Ile Ile Gly Gly 210 215 220 Cys AlaLeu Ala Ala Val Thr Glu Leu Asn Phe Asn Met Val Gly Phe 225 230 235 240Met Gly Ala Met Ile Ser Asn Leu Ala Phe Val Phe Arg Asn Ile Phe 245 250255 Ser Lys Arg Gly Met Lys Gly Lys Ser Val Ser Gly Met Asn Tyr Tyr 260265 270 Ala Cys Leu Ser Ile Met Ser Leu Val Ile Leu Thr Pro Phe Ala Ile275 280 285 Ala Met Glu Gly Pro Gln Met Trp Ala Ala Gly Trp Gln Lys AlaLeu 290 295 300 Ala Glu Val Gly Pro Asn Val Val Trp Trp Ile Ala Ala GlnSer Val 305 310 315 320 Phe Tyr His Leu Tyr Asn Gln Val Ser Tyr Met SerLeu Asp Gln Ile 325 330 335 Ser Pro Leu Thr Phe Ser Ile Gly Asn Thr MetLys Arg Ile Ser Val 340 345 350 Ile Val Ser Ser Ile Ile Ile Phe His ThrPro Val Arg Ala Val Asn 355 360 365 Ala Leu Gly Ala Ala Ile Ala Ile LeuGly Thr Phe Leu Tyr Ser Gln 370 375 380 Ala Lys Ala 385

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide havingglucose-6-phosphate/phosphate translocator activity, wherein the aminoacid sequence of the polypeptide and the amino acid sequence of SEQ IDNO:4 have at least 86% sequence identity based on the Clustal alignmentmethod, or (b) the complement of the nucleotide sequence, wherein thecomplement and the nucleotide sequence contain the same number ofnucleotides and are 100% complementary.
 2. The polynucleotide of claim1, wherein the amino acid sequence of the polypeptide and the amino acidsequence of SEQ ID NO:4 have at least 90% sequence identity based on theClustal alignment method.
 3. The polynucleotide of claim 1, wherein theamino acid sequence of the polypeptide and the amino acid sequence ofSEQ ID NO:4 have at least 95% sequence identity based on the Clustalalignment method.
 4. The polynucleotide of claim 1, wherein thenucleotide sequence comprises the nucleotide sequence of SEQ ID NO:3. 5.The polynucleotide of claim 1, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:4.
 6. A recombinant DNA constructcomprising the polynucleotide of claim 1 operably linked to a regulatorysequence.
 7. A method for transforming a cell comprising transforming acell with the polynucleotide of claim
 1. 8. A cell comprising therecombinant DNA construct of claim
 6. 9. A method for producing a plantcomprising transforming a plant cell with the polynucleotide of claim 1and regenerating a plant from the transformed plant cell.
 10. A plantcomprising the recombinant DNA construct of claim
 1. 11. A seedcomprising the recombinant DNA construct of claim
 1. 12. A vectorcomprising the polynucleotide of claim 1.